This is an application for an extension of MERIT award R37HL056182, beyond the original 5 year funding period. Here we present a progress report regarding the first four years of the project. The overall goal of this project is to understand how the BMP signaling pathway regulates hematopoiesis. We made significant progress toward this goal, resulting so far in 15 peer-reviewed original research articles. The original Aims of the proposal were to 1) Define the spatio-temporal requirements of Smad signaling for hemato-vascular development; 2) Delineate the specificity of Smadi and Smad5 for hematopoiesis; and 3) Determine the molecular mechanisms by which Smad function regulates hematopoiesis. We request funding for an additional 5 years of support, to continue this project pursuing these same Aims. Our studies have revealed considerable complexity in the specificity of BMP signaling, and indicate that much more remains to be discovered regarding downstream components, interacting pathways, and molecular mechanisms. There has been substantial technology development that allows new experimental strategies, primarily based on chemical biology, deep-sequencing, and proteomics. Therefore, our objectives remain the same, while experimental strategies have evolved. Specifically, we propose to 1) Use our zebrafish transgenic models and known or newly discovered small molecule compounds to modulate BMP signaling, and define stage-specific functions. Network analysis of altered pathway components will be used to identify new components that will be tested for function in the pathway, and for hematopoiesis; 2) Use our novel murine ESC models to develop a comprehensive description, at the whole genome level, of alterations in transcript profiles and epigenetic patterns controlled by Smadi at either an early stage (expanding progenitors) or a later stage (restricting progenitors). Key targets will be defined by network analysis and tested for function by forced expression or knockdown; and 3) Generate a comprehensive description for gene regulation mediated by BMP signaling that functions at the post-transcriptional level. This has the potential to identify entirely novel regulatory mechanisms.